Myc oncoproteins (c-, N-, and L-Myc) are transcription factors, which are involved in the pathogenesis of many human cancers. In addition to being transforming, c-Myc, the most intensely studied member also promotes apoptosis, alters morphology, inhibits differentiation, accelerates cell cycle progression, and promotes genomic instability. A major goal is to identify the genes regulated by c-Myc, since this would provide significant insight into the molecular basis of Myc's phenotypes. Another goal is to determine whether different Myc members regulate the same target genes. Several c-Myc target genes can mimic a limited number of c-Myc properties. However, the cells in which they have been shown to do so all express endogenous c-Myc, thus making it impossible to determine whether these targets behave in a truly c-Myc-independent manner. We have identified a c-Myc target gene, MT-MC1, which has the unique property of imparting multiple c-Myc phenotypes in cells engineered to not express c-Myc (c-Myc "K.O" cells). This suggests that MT-MC1, and a carefully selected group of other complementing genes, might be used to reconstruct the c-Myc phenotype in KO cells, thus defining a minimum, although not necessarily unique, functional target gene population. We have therefore devised a novel retroviral vector (pRetroFLOX-GFP) that permits an unlimited number of genes to be sequentially transduced and stably expressed. This will allow us in Specific Aim 1, to construct and characterize a series of pRetroFLOX-GFP vectors for a defined subset of c-Myc target genes. In Specific Aim 2, we will determine whether over-expression of individual c-Myc target genes in KO cells can mimic the same c-Myc phenotypes as they do in parental cells. In Specific Aim 3, we will consecutively express c-Myc target genes in KO cells or transgenic mice in order to recapitulate the "complete" c-Myc phenotype. In related studies, we have shown that the CCL6 chemokine is regulated oppositely by c-Myc and L-Myc. Together with c-Myc, CCL6 imparts growth factor independence and a transformed phenotype to IL-3-dependent myeloid cells and enhances the invasive and metastatic behavior of established tumor lines. This appears to be a result of CCL6's ability to induce apoptotic death in adjacent normal cells, thus destroying the normal tissue barriers that limit tumor spread. Therefore, in Specific Aim 4, we will determine whether CCL6 must be secreted in order to impart IL-3-independent growth and more aggressive tumor behavior. In Specific Aim 5, we will determine the mechanism(s) by which CCL6 and c-Myc subvert the IL-3 signaling pathway. In Specific Aim 6, we will determine whether related chemokines impart CCL6-like properties. Finally, in Specific Aim 7, we will develop a transgenic model of CCL-6-dependent tumor invasion. Together, these studies will provide new insights into the mechanisms by which c-Myc and its transcriptional targets subvert normal cellular pathways en route to establishing a transformed cell.